UBASH3A Antibody, HRP conjugated

What is UBASH3A and why is it important in immunological research?

UBASH3A is a multidomain protein containing N-terminal UBA (ubiquitin-associated), SH3 (Src homology 3), and C-terminal histidine phosphatase (PGM) domains. It plays a significant role in T-cell receptor signaling as a negative regulator . The importance of UBASH3A in immunological research stems from its involvement in autoimmune diseases, particularly type 1 diabetes, where disease-associated genetic variants act by increasing UBASH3A expression in human primary T cells, leading to reduced IL-2 production upon TCR stimulation . Understanding UBASH3A function provides critical insights into T-cell activation regulation and autoimmune disease mechanisms, making antibodies against this protein valuable research tools for investigating these pathways.

What applications are UBASH3A antibodies suitable for?

Based on current research, UBASH3A antibodies have been validated for several experimental applications:

• Western Blotting (WB): For detecting UBASH3A protein in cell lysates

• Immunohistochemistry on paraffin-embedded sections (IHC-P): For tissue localization studies

• Immunocytochemistry/Immunofluorescence (ICC/IF): For cellular localization studies, as demonstrated in human bone marrow erythroleukemia cell lines

• Immunoprecipitation (IP): For protein-protein interaction studies as shown in co-immunoprecipitation experiments with CBL-B and other interacting partners

HRP-conjugated UBASH3A antibodies would be particularly useful for Western blotting and ELISA applications where the enzymatic activity of HRP provides signal amplification for enhanced detection sensitivity.

How do I optimize Western blotting protocols for UBASH3A detection using HRP-conjugated antibodies?

For optimal Western blotting results with HRP-conjugated UBASH3A antibodies:

• Sample preparation: Prepare whole-cell lysates using NETN buffer (20 mM Tris-HCl, pH 8.0, 0.6 M NaCl, 0.5% NP-40, and 1 mM EDTA) with protease inhibitors .

• Gel electrophoresis: Use NuPAGE Tris-Acetate or Bolt Bis-Tris Plus gels for optimal resolution of UBASH3A (~70 kDa) .

• Protein transfer: Transfer to PVDF membranes, which provide better protein retention and signal-to-noise ratio for HRP detection.

• Blocking: Block membranes with 5% non-fat dry milk in TBST for 1 hour at room temperature to reduce non-specific binding.

• Antibody dilution: Start with a 1:1000 dilution for primary antibody incubation (overnight at 4°C), though optimization may be required based on the specific antibody's titer.

• Washing: Perform multiple TBST washes (3-5 times, 5 minutes each) between steps to reduce background.

• Detection: Use appropriate chemiluminescent substrates compatible with HRP for signal development. Scanning can be performed using instruments like the Typhoon 9200 laser scanner with quantification using ImageQuant TL software .

• Controls: Include positive controls (Jurkat cell lysates) and negative controls (UBASH3A−/− cell lysates) as demonstrated in published research .

What are the structural domains of UBASH3A and how do they affect antibody selection?

UBASH3A contains three key structural domains that influence antibody selection considerations:

When selecting an UBASH3A antibody, consider which domain you want to target based on your research question. For general detection, antibodies targeting recombinant fragments within amino acids 100-350 or 250-400 have been validated . For studying specific interactions or conformational states, domain-specific antibodies may be more appropriate.

How can I distinguish between UBASH3A and its paralog UBASH3B in experimental systems?

Distinguishing between UBASH3A and UBASH3B is crucial for accurate experimental interpretation due to their structural similarities but functional differences:

• Antibody specificity: Select antibodies raised against regions with lowest sequence homology between the paralogs. Commercial antibodies such as ab197168 and ab251834 are specifically validated against UBASH3A .

• Functional validation: Unlike UBASH3B, UBASH3A exhibits negligible protein tyrosine phosphatase activity at neutral pH . Design functional assays that exploit this difference, such as phosphatase activity measurements.

• Expression patterns: UBASH3A and UBASH3B show different tissue and cell-type expression patterns. Validate by qPCR using paralog-specific primers.

• Knockout controls: Include UBASH3A−/− cells (generated via CRISPR/Cas9 as described in the literature) as negative controls to confirm antibody specificity .

• Molecular weight differences: Although similar, the paralogs may show slight differences in molecular weight or post-translational modification patterns on Western blots.

• Immunoprecipitation-mass spectrometry: For definitive identification, immunoprecipitate with paralog-specific antibodies followed by mass spectrometry confirmation of unique peptide sequences.

What methods can be used to study UBASH3A interactions with CBL-B and how can HRP-conjugated antibodies facilitate these studies?

UBASH3A interaction with CBL-B is physiologically significant for T-cell activation regulation. Here are methods to study this interaction:

• Co-immunoprecipitation (Co-IP): The SH3 domain of UBASH3A mediates binding to CBL-B . For Co-IP:

  • Pre-clear whole-cell lysates from cells expressing both proteins

  • Immunoprecipitate with anti-UBASH3A antibody (10 μg per 10 mg lysate)

  • Include UBASH3A−/− lysates as negative controls

  • Detect with anti-CBL-B antibody (clone G-1, Santa Cruz)

  • HRP-conjugated secondary antibodies provide enhanced sensitivity for detecting co-immunoprecipitated proteins

• Proximity Ligation Assay (PLA):

  • Use specific primary antibodies against UBASH3A and CBL-B

  • Secondary antibodies with conjugated oligonucleotides enable visualization of protein interactions in situ

  • HRP-conjugated detection systems can be employed for chromogenic visualization

• Domain mapping:

  • Generate UBASH3A constructs with SH3 domain mutations (e.g., W279A)

  • Compare wild-type and mutant binding to CBL-B

  • Detect interactions using HRP-conjugated antibodies in Western blot analysis

• Functional assays:

  • Assess how manipulating UBASH3A levels affects CBL-B-mediated functions

  • Monitor T-cell activation markers with flow cytometry

  • Use HRP-conjugated antibodies for intracellular signaling studies

How can post-translational modifications of UBASH3A be detected and what impact do they have on antibody recognition?

UBASH3A undergoes several post-translational modifications that influence its function and potentially affect antibody recognition:

• Ubiquitination: UBASH3A has four identified ubiquitination sites at lysine residues 15, 202, 309, and 358 . Monoubiquitination at K202 causes conformational changes that prevent UBA domain interaction with substrates.

  Detection methods:

  • Immunoprecipitate UBASH3A and probe with anti-ubiquitin antibodies

  • Use K202R mutants as controls

  • HRP-conjugated secondary antibodies provide sensitive detection of ubiquitinated species

  Impact on antibody recognition: Antibodies targeting regions near ubiquitination sites may show differential binding to modified versus unmodified UBASH3A. Consider using antibodies raised against epitopes distant from these sites for consistent detection.

• Phosphorylation: Although UBASH3A has weak phosphatase activity, it may itself be phosphorylated during T-cell activation.

  Detection methods:

  • Phospho-specific antibodies (if available)

  • Phosphoprotein staining following gel electrophoresis

  • Mass spectrometry analysis of immunoprecipitated UBASH3A

  Impact on antibody recognition: Phosphorylation can mask epitopes or alter protein conformation, potentially affecting antibody binding. Test antibody reactivity under different cell stimulation conditions.

What experimental approaches can distinguish between the different functional roles of UBASH3A in T-cell receptor regulation?

UBASH3A impacts T-cell receptor regulation through multiple mechanisms. To distinguish between these roles:

• TCR-CD3 synthesis regulation:

  • Compare total cellular CD3 chain levels in UBASH3A−/− versus wild-type or overexpressing cells

  • Use Western blotting with HRP-conjugated antibodies for quantification

  • Normalize to housekeeping proteins like GAPDH

  • Pulse-chase experiments with metabolic labeling to track newly synthesized TCR-CD3

• Cell-surface TCR-CD3 downmodulation:

  • Flow cytometry with anti-CD3ε antibodies at various time points following TCR engagement

  • Compare downmodulation kinetics between UBASH3A−/− cells (e.g., clone 2.1F7) and UBASH3A-overexpressing cells (e.g., clone 2F5)

  • For the protocol:

    • Incubate cells with APC-conjugated anti-CD3ε (2.5 μg/mL)

    • Measure internalization at time points from 0 to 60 minutes

    • Use acid wash (100 mM glycine, 100 mM NaCl, pH 2.5) to remove surface-bound antibodies

• Interaction with endocytic machinery:

  • Co-immunoprecipitation studies with dynamin

  • Dynamin inhibition experiments to assess UBASH3A-dependent effects

  • Live-cell imaging of fluorescently tagged UBASH3A and endocytic components

• Association with ERAD components:

  • Mass spectrometry analysis of UBASH3A interactome

  • Co-localization studies using confocal microscopy

  • Functional assays measuring ER-associated degradation of TCR components

What are the optimal fixation and staining protocols for detecting UBASH3A in tissue and cell samples using HRP-conjugated antibodies?

For optimal immunohistochemical and immunocytochemical detection of UBASH3A:

• Cell fixation:

  • For immunofluorescence: PFA fixation (4%) with Triton X-100 (0.1%) permeabilization has been validated for UBASH3A detection in human cell lines

  • For tissue sections: Formalin fixation followed by paraffin embedding is compatible with UBASH3A antibodies

• Antigen retrieval (for paraffin sections):

  • Heat-induced epitope retrieval using citrate buffer (pH 6.0)

  • Optimization may be needed depending on tissue type and fixation duration

• Blocking:

  • Use 5-10% normal serum from the same species as the secondary antibody

  • Include 0.1-0.3% Triton X-100 for permeabilization if detecting intracellular epitopes

• Primary antibody incubation:

  • Tested concentration: 4 μg/ml for ICC/IF applications

  • Incubate overnight at 4°C for optimal sensitivity

  • Include appropriate negative controls (isotype control or UBASH3A knockout samples)

• HRP detection systems:

  • For direct HRP-conjugated UBASH3A antibodies: Apply directly after blocking

  • For indirect detection: Use HRP-conjugated secondary antibodies

  • Develop with DAB (3,3'-diaminobenzidine) for bright-field microscopy

  • Amplification systems like tyramide signal amplification (TSA) can enhance sensitivity

• Counterstaining:

  • Hematoxylin for brightfield

  • DAPI or Hoechst for fluorescence to visualize nuclei

How can UBASH3A antibodies be utilized to study its role in autoimmune conditions, particularly type 1 diabetes?

UBASH3A genetic variants have been associated with type 1 diabetes (T1D), with disease-associated variants increasing UBASH3A expression in primary T cells . Researchers can utilize UBASH3A antibodies to:

• Expression analysis in patient samples:

  • Compare UBASH3A protein levels in peripheral blood mononuclear cells (PBMCs) from T1D patients versus healthy controls

  • Correlate expression with genotype at disease-associated loci

  • Use Western blotting with HRP-conjugated antibodies for quantitative analysis

• Functional studies in disease models:

  • Assess how UBASH3A expression affects T-cell activation in response to autoantigens

  • Measure IL-2 production (which is reduced with increased UBASH3A expression)

  • Track TCR-CD3 dynamics in T cells with different UBASH3A expression levels

• Histological analysis of pancreatic tissue:

  • Compare UBASH3A expression in infiltrating T cells in pancreatic islets

  • Double staining with islet cell markers to study proximity to target cells

  • Use HRP-conjugated detection systems for sensitive visualization

• Mechanistic investigations:

  • Study UBASH3A interaction with other autoimmunity-associated proteins

  • Investigate how disease-associated variants affect protein-protein interactions

  • Analyze post-translational modifications in disease contexts

What experimental considerations are important when comparing UBASH3A expression and function between different T-cell subsets?

T-cell subsets exhibit functional heterogeneity that may influence UBASH3A expression and function. Important considerations include:

• Subset isolation and purity:

  • Use magnetic bead isolation or fluorescence-activated cell sorting for high-purity populations

  • Confirm subset identity with canonical markers (CD4+, CD8+, Tregs, Th1, Th2, etc.)

  • Include purity checks in experimental documentation

• Basal expression analysis:

  • Compare UBASH3A levels across subsets using:

    • Western blotting with HRP-conjugated detection systems

    • Flow cytometry for single-cell resolution

    • qRT-PCR for transcriptional analysis

  • Normalize to appropriate housekeeping genes/proteins for each subset

• Activation-induced changes:

  • Monitor UBASH3A expression changes following:

    • TCR stimulation (anti-CD3/CD28)

    • Cytokine treatment

    • Co-stimulatory/inhibitory signals

  • Establish time courses to capture dynamic responses

• Functional readouts:

  • Subset-specific functions (cytokine production, proliferation, cytotoxicity)

  • TCR-CD3 downmodulation kinetics

  • Interaction with subset-specific signaling components

• In vivo relevance:

  • Compare findings in primary cells versus cell lines

  • Consider tissue-resident versus circulating T-cell populations

  • Assess relevance to disease-specific T-cell abnormalities

What are common sources of non-specific binding with UBASH3A antibodies and how can these be mitigated?

Non-specific binding can complicate UBASH3A detection. Common sources and mitigation strategies include:

• Cross-reactivity with related proteins:

  • UBASH3A shares structural similarities with its paralog UBASH3B

  • Validate specificity using UBASH3A−/− controls generated via CRISPR/Cas9

  • Consider pre-absorption of antibodies with recombinant UBASH3B

• Inadequate blocking:

  • Increase blocking agent concentration (5-10% normal serum or BSA)

  • Extend blocking time (1-2 hours at room temperature)

  • Consider alternative blocking agents if background persists

• Secondary antibody cross-reactivity:

  • Use secondary antibodies pre-adsorbed against species present in your samples

  • Include secondary-only controls in all experiments

  • For HRP-conjugated primaries, include isotype controls

• Sample preparation issues:

  • Incomplete cell/tissue lysis may cause aggregates that bind antibodies non-specifically

  • Centrifuge lysates at high speed to remove particulates

  • Filter samples if necessary

• Detection system sensitivity:

  • HRP can be highly sensitive, potentially amplifying background

  • Titrate antibody concentrations to find optimal signal-to-noise ratio

  • Reduce substrate incubation time if background is high

• Fixation artifacts:

  • Overfixation can increase non-specific binding

  • Optimize fixation times and conditions

  • Include appropriate antigen retrieval steps

How can researchers troubleshoot inconsistent results when using UBASH3A antibodies for monitoring T-cell activation?

When studying UBASH3A in T-cell activation contexts, several factors can lead to inconsistent results:

• Heterogeneous activation states:

  • Standardize activation protocols (concentration and timing of stimuli)

  • Use flow cytometry to confirm activation status with markers like CD69

  • Consider single-cell approaches to account for cellular heterogeneity

• Temporal dynamics:

  • UBASH3A's role in TCR-CD3 dynamics is time-dependent

  • Establish detailed time courses (0-60 minutes after stimulation)

  • Synchronize cell populations before stimulation (serum starvation or resting)

• Technical variability in TCR-CD3 downmodulation assays:

  • Standardize acid wash protocols for removing surface-bound antibodies

  • Include both acid-washed and non-acid-washed controls

  • Maintain consistent temperature during internalization (37°C)

• Expression level variations:

  • Compare results between wild-type, knockout, and overexpression systems

  • Quantify UBASH3A levels in each experimental condition

  • Normalize functional readouts to expression levels

• Experimental system differences:

  • Jurkat cells versus primary T cells may show different UBASH3A functions

  • Account for donor variability in primary cell experiments

  • Consider genetic background when using modified cell lines

How can UBASH3A antibodies be incorporated into high-throughput or multiplexed detection systems?

Emerging technologies enable higher-throughput analysis of UBASH3A in complex biological systems:

• Multiplexed immunoassays:

  • Bead-based platforms (e.g., Luminex) allow simultaneous detection of UBASH3A and interacting partners

  • Microwestern arrays for analyzing multiple signaling proteins in limited samples

  • HRP-conjugated antibodies can be used with appropriate detection substrates compatible with multiplexed systems

• Mass cytometry (CyTOF):

  • Metal-tagged UBASH3A antibodies enable single-cell analysis alongside dozens of other markers

  • Particularly valuable for heterogeneous samples like PBMCs from patients with autoimmune conditions

  • Allows correlation of UBASH3A levels with cell subset identity and activation status

• Single-cell western blotting:

  • Analyzes UBASH3A expression at single-cell resolution

  • Captures cell-to-cell variability masked in conventional western blots

  • HRP detection systems compatible with microfluidic platforms

• Tissue microarray analysis:

  • High-throughput analysis of UBASH3A expression across multiple tissue samples

  • Particularly valuable for comparative studies of different autoimmune conditions

  • HRP-based chromogenic or fluorescent detection systems applicable

• Automated immunohistochemistry platforms:

  • Standardized staining protocols ensure reproducibility

  • High-throughput processing of multiple samples

  • Digital pathology analysis for quantitative assessment

What methodological approaches can be used to study the dynamic interaction between UBASH3A and the TCR-CD3 complex in live cells?

Understanding the dynamic interactions between UBASH3A and TCR-CD3 requires sophisticated live-cell approaches:

• Förster Resonance Energy Transfer (FRET):

  • Generate fluorescently tagged UBASH3A and CD3 components

  • Monitor protein-protein interactions in real-time

  • Requires careful controls to validate proximity vs. direct interaction

• Fluorescence Recovery After Photobleaching (FRAP):

  • Track mobility of fluorescently tagged UBASH3A

  • Compare dynamics in resting vs. activated T cells

  • Assess how mutations in functional domains affect mobility

• Total Internal Reflection Fluorescence (TIRF) microscopy:

  • Visualize UBASH3A recruitment to the immunological synapse

  • Monitor interactions with TCR-CD3 complexes at the plasma membrane

  • High spatial resolution at the cell-substrate interface

• Lattice light-sheet microscopy:

  • Capture 3D dynamics with minimal phototoxicity

  • Follow UBASH3A trafficking during T-cell activation

  • Combine with super-resolution techniques for enhanced detail

• Optogenetic approaches:

  • Use light-inducible dimerization to manipulate UBASH3A localization

  • Assess functional consequences of forced interactions with TCR-CD3

  • Combine with live-cell reporters of T-cell activation

• Quantitative image analysis:

  • Track co-localization coefficients over time

  • Measure recruitment/dissociation kinetics

  • Correlate with functional readouts of TCR signaling

While these techniques typically use fluorescent proteins or tags rather than HRP-conjugated antibodies, they provide complementary information to biochemical approaches using HRP detection systems.